Expression of LIM domain-binding 3 (LDB3), a striated muscle Z-band alternatively spliced PDZ-motif protein in the nervous system

LIM domain-binding 3 (LDB3) is a member of the Enigma family of PDZ–LIM proteins. LDB3 has been reported as a striated muscle-specific Z-band alternatively spliced protein that plays an important role in mechanosensory actin cytoskeleton remodeling. This study shows that LDB3 is broadly expressed in the central and peripheral nervous system of human and mouse. LDB3 is predominantly expressed in the adult stages compared to early development and at a significantly higher level in the spinal cord than in the brain. As in skeletal muscle and heart, LDB3 is extensively alternatively spliced in the neurons. Three novel splice isoforms were identified suggesting splicing-dependent regulation of LDB3 expression in the nervous system. Expression of LDB3 in the motor cortex, cerebellum, spinal motor neuron, peripheral nerve, and neuromuscular junction in addition to skeletal muscle indicates important roles for this PDZ–LIM family protein in motor planning and execution. Moreover, expression in the hippocampal neurons suggests roles for LDB3 in learning and memory. LDB3 interactors filamin C and myotilin are also expressed in the spinal motor neuron, nerve, and neuromuscular junction, thereby providing the basis for neurogenic manifestations in myopathies associated with mutations in these so-called muscle proteins.


Results
Expression of LDB3 in mouse and human brain and spinal cord. Total cellular Ldb3 mRNA levels measured by ddPCR were increased 19-fold and 16-fold, respectively from postnatal day 10 to age six months in mouse brain and spinal cord (Fig. 1A). Compared to brain, Ldb3 mRNA levels were tenfold higher in the spinal cords of six month old mice ( Fig. 1A; Bonferroni adjusted p < 0.0001). Immunoblotting of human brain and spinal cord lysates showed higher levels of LDB3 protein in spinal cord relative to brain (Fig. 1B).
LDB3 splice isoforms in mouse and human brain and spinal cord. We consistently observed multiple bands in the immunoblots of brain and spinal cord lysates similar to that seen in muscle 4 , suggesting the presence of alternatively spliced LDB3 isoforms in the nervous system. Higher yield of total cellular RNA with RNA Integrity Number (RIN) > 9 enabled examination of the LDB3 splice isoforms with Single Molecule Real Time (SMRT) sequencing in the brain of 6 month old mice. Novel Ldb3 splice isoforms containing exons 3-6-8 cassette were identified in long and short Ldb3 isoform libraries ( Fig. 1C; Ldb3-L and Ldb3-S, respectively; Genbank ID MH027380-MH027382). A majority of reads conformed to the annotated Ldb3 transcripts on the Ensemble genome browser and contained exon 4 in Ldb3-L and Ldb3-S libraries. The exons 5-6-7 cassette was detected only rarely in the brain. Ldb3 exons 4 and 6 were mutually exclusive, supporting previous observations in skeletal muscle and heart 6,7,24 . About two-thirds of LDB3-L reads excluded exon 11 (exon 10 in human). Next, we validated the SMRT sequencing findings with RT-PCR, Sanger sequencing, and immunofluorescence assays.
RT-PCR followed by Sanger sequencing of cloned amplicons generated using primers in canonically spliced exons 3 and 8 validated the exon 3-4-8 splicing similar to that seen in heart, the exon 3-5-6-7-8 splicing as in skeletal muscle, and novel exon 3-6-8 splicing in brain and spinal cord of 6 month old mice (Fig. 1D). LDB3 exon 3-6-8 splicing was not detected in heart or skeletal muscle. We confirmed both exon 11 (exon 10 in human) inclusion and exclusion in Ldb3-L isoforms in brain and spinal cord by sequencing of RT-PCR products using primers in canonically spliced exons 8 and 13 (Fig. 1D). This splicing pattern was similar to heart but contrasts with skeletal muscle in which most Ldb3-L isoforms excluded exon 11 (Fig. 1D).
Expression of distinct LDB3 isoforms was also examined by immunostaining with custom-made rabbit polyclonal antibodies against epitopes in alternatively spliced LDB3 exons exon 6 23 , 4 and 9 (exon 10 in mice; Supplementary Fig. 1A). These knockout validated antibodies specifically recognized corresponding LDB3 exonencoded domains in recombinant purified LDB3 peptides, transfected cell culture systems, and in mouse vastus lateralis muscle and heart (Supplementary Fig. 1B-E 23 ). Immunofluorescent staining with these validated antibodies showed LDB3 isoforms containing exon 4-,6-and 10-encoded domains in the cell bodies and proximal processes of anterior horn neurons in the spinal cord of 6 month old mice (Fig. 1E). Further, immunohistochemical staining with these antibodies detected distinct LDB3 isoforms in the nucleus dorsalis (Clarke's nucleus) neurons in the posterior horn, preganglionic sympathetic neurons in the lateral horn, and the anterior horn neurons in the human spinal cord L1-L2 sections (Fig. 1F).
Spatial expression of LDB3 in the mouse brain. We examined LDB3 expression in functionally different brain regions including the motor cortex, hippocampus, and cerebellum of 6 month old mice. For this purpose we used previously published LDB3 antibodies in immunofluorescence assays 8,23,25 . These antibodies recognized both LDB3-L and LDB3-S isoforms in immunoblotting assays ( Supplementary Fig. 1B,C). LDB3 immunostaining was detected in the soma and dendrites of neurons in the motor cortex layers II, III, and V ( Fig. 2A). The target specificity of the antibody was confirmed by staining the vastus lateralis muscle of Ldb3 +/+ and Ldb3 −/− littermates on the same slide as the brain tissues (Fig. 2B). We also found LDB3 in the CA1 pyramidal neurons of the hippocampus (Fig. 2C). In the cerebellar cortex, LDB3 staining was mostly localized to the dendritic arbors of Purkinje cells (Fig. 2D). LDB3 staining was absent after blocking the antibody by incubating with purified untagged LDB3 peptide, thus validating the antibody specificity for LDB3 protein in neurons.
LDB3 expression in human motor cortex and spinal cord. Next, we examined the expression of LDB3 in coronal sections of human motor cortex and spinal cord L1-L2 axial sections from autopsy by immunostaining with a knockout validated antibody. Immunohistochemical staining detected LDB3 in neurons from layers II to VI of the human motor cortex (Fig. 3A). As in mice, LDB3 immunoreactivity was seen in the soma and processes of neurons in layers II, III, and V (Fig. 3B). Immunofluorescence studies confirmed the presence of LDB3 in the MAP2-stained cell body and dendrites of the pyramidal neurons including the gigantic Betz cells in layer V of the human motor cortex (Fig. 3C). In L1-L2 human spinal cord, LDB3 immunoreactivity was seen in the soma and processes of the anterior horn neurons (Fig. 3D).

Expression of LDB3 and its interactors filamin C and myotilin in the peripheral nervous system.
In the anterior horn of the spinal cord, cholinergic motor neurons are unique neuronal cells connecting the brain to the periphery through long axons terminating in neuromuscular junctions (NMJs) in skeletal muscles. Lower motor neuron syndrome and axonal neuropathy have been reported in patients with mutations in LDB3, filamin C, and myotilin 16,17,19 . We asked whether LDB3 and its interacting proteins filamin C and myotilin are expressed in the cholinergic motor neurons of the spinal cord, peripheral nerves, and NMJs. We detected LDB3 in the soma and processes of ChAT positive motor neurons in the anterior horn of the spinal cord from 6 month old mice (Fig. 4A). The antibody signal was absent in the sections incubated with blocking peptide as in the brain. Staining of parallel spinal cord sections showed filamin C and myotilin immunoreactivity in the ChAT positive motor neurons (Fig. 4B,C). As with LDB3 (Fig. 3D), immunohistochemical assays detected filamin C and myotilin antibody stained anterior horn neurons in human L1-L2 spinal cord axial sections (Fig. 4D). www.nature.com/scientificreports/ www.nature.com/scientificreports/ LDB3, filamin C, and myotilin were detected in intramuscular nerves and NMJs in the tibialis anterior muscle of six month old mice (Fig. 5A,B).

Discussion
LDB3 has thus far been regarded as a muscle-specific protein that plays an important role in maintaining the actin cytoskeleton integrity in multiple species [26][27][28] . The present study demonstrates the developmental and spatial expression of LDB3 in the mouse and human nervous system. The precedence for expression of a presumed muscle-specific LIM protein in the nervous system was established by Muscle LIM Protein which was subsequently shown to promote neuroregeneration 29,30 . Similar to muscle, multiple splice isoforms of LDB3 exist in the nervous system including three as yet unidentified isoforms. LDB3 is predominantly expressed in the cell body and dendrites of neurons during adult stages of mouse brain and spinal cord. Relative to brain, LDB3 expression levels are significantly higher in spinal cord. In particular LDB3 is expressed in the upper motor neurons, cholinergic lower motor neurons, peripheral nerve, and NMJ that effectively regulate muscle contraction. Moreover, we found that LDB3 is expressed in the cerebellar Purkinje cells regulating coordination and learning of movements, and the hippocampal CA1 pyramidal neurons fundamental to learning and formation of memories.
In terms of anatomical localization and agility, the vulnerability of spinal cord to undergo a greater mechanical stress as compared to brain may require more frequent repair of cytoskeletal proteins for stabilization of cellular function 31 . Increased reactive oxygen species production in mitochondria of spinal cord as compared to brain leads to greater propensity of oxidative stress in motor neurons of the spinal cord 32 . There is in-vitro evidence that most neuronal and glial cell types adapt their morphology and cytoskeletal configuration to mechanical stimuli in their environment 33 . LDB3 is a cytoskeleton associated protein and binds to mechanosensitive region of cytoskeletal proteins linked to actin and some known heat shock proteins (HSPs) thus functions as a multivalent interaction hub contributing in cellular mechanosensing 23 . In this background, the underlying reason for a significantly high LDB3 expression in spinal cord compared to the brain in adult mice could be a cellular counterintuitive strategy to deal with greater mechanical stress in spinal cord. Like our findings, a significantly higher expression of HSPs also interact and co-operate with LDB3 23 , was reported in spinal cord as compared to brain in wild type mice and in multiple sclerosis patient tissue 31,34 . HSPs interact with the cytoskeleton to avoid aggregation, support assembly, and facilitate stabilization of cytoskeletal proteins. The underlying reason for the high HSPs was attributed to differences in astrocyte cytoskeleton composition and to a subset of astrocytes and microglia with higher HSP expression in spinal cord and cortical primary mixed glial cultures 31,34 . Broader We applied long-read sequencing technology to map alternative splice sites within single Ldb3-L and Ldb3-S transcripts in the nervous system. A large majority of isoforms in the mouse nervous system can be explained by alternative splicing at sites previously reported in studies of heart and skeletal muscle 6,7,24 . In mouse, Ldb3 isoforms with exon 4 were known to be expressed only in the heart 7 . Here, we show that the Ldb3-L and Ldb3-S isoforms with exon 4 are also predominent in brain and spinal cord. In contrast, skeletal muscle-specific Ldb3 isoforms containing the exon 5-6-7 cassette were only rarely detected in the brain. The novel Ldb3 splice isoforms with the exon 3-6-8 cassette in the brain and spinal cord were absent in skeletal muscle and heart. Nonsensemediated mRNA decay likely affected the transcript levels as joining of exon 3 to 6 shifts the reading frame resulting in a premature termination codon within exon 6. We generated and validated new domain-specific antibodies which identified specific LDB3 isoforms in mouse and human skeletal muscle, heart, and neurons. These antibodies will be useful in studies of LDB3 isoform-specific roles in different tissues. RT-PCR assays and immunofluorescence with isoform-specific antibodies detected expression of LDB3 exon 4, 6, and 10 (exon 9 in human) in neurons, which contain hotspots for rare mutations causing skeletal and cardiac myopathy 17,24 . In addition to LDB3, other rare myopathy gene products such as filamin C and myotilin are expressed in the nervous system [this study; (Mologni 13 ; Xie 14 )]. Clinical studies are needed to better define the natural history and full spectrum of disease presentation in patients with LDB3, filamin C, and myotilin mutations.
Multiple LDB3 splice isoforms enable heterogeneous protein interactions, modify mutation effects and alter disease severity 8,23,35 . The PDZ domain is present in all LDB3 isoforms, whereas LIM domains are only present in www.nature.com/scientificreports/ Ldb3-L splice isoforms 6,7,24 . We identified both Ldb3-L and Ldb3-S isoforms in the nervous system. The Ldb3-L isoforms negatively regulate PKCα expression 10 . LDB3 avidly binds to actin and facilitates mechanosensingdirected actin cytoskeleton remodeling 8,9,23 . As an A-kinase anchoring protein, LDB3 plays a role in β-adrenergic stimulation of cardiac Ca v 1.2 channels 36 . Interestingly, the spatial distribution of LDB3 mirrored that of MAP2, a dominant A-kinase anchoring protein in neurons 37 . These LDB3 functions are not unlike other PDZ scaffold proteins, for example postsynaptic density protein 95, which facilitate functional coupling of receptors and ion channels to downstream signaling pathways in synapses and neurons 38 . More recent studies indicate the role of activity dependent actin cytoskeleton remodeling in synapse-specific delivery and plasticity 39 . Whereas LDB3 is detectable in myocytes from early embryonic stages to adulthood 5 , we found that Ldb3 expression in the mouse nervous system occurs preferentially during adult stages. In agreement with previous observations 28 , LDB3deficient mice did not survive after birth which limited their use to examine the role of LDB3 in the adult nervous system. While outside the scope of this study, conditional gene knockout model systems are necessary to solve the enigma of LDB3 functions in the central and peripheral nervous system during specific stages of development.

Conclusion
In summary, our findings indicate that LDB3 is expressed broadly in the adult central and peripheral nervous system of mouse and human. The splicing pattern of LDB3 in the brain and spinal cord is more similar to that of heart than skeletal muscle. The expression of LDB3 and interacting proteins in the nervous system indicates expansion of clinical phenotype and genotype relationship in degenerative diseases. Importantly, LDB3 has roles in regulating activity-dependent actin cytoskeleton remodeling and its presence in the neurons should stimulate investigations for novel mechanisms of neuronal trafficking and synaptic plasticity.

Materials and methods
Mice. Wild   www.nature.com/scientificreports/ Antibodies. Primary antibodies used in this study are in Supplementary Table 1 (Supplementary file 1).
Alexa Fluor-and HRP-conjugated secondary antibodies were purchased from Invitrogen and Jackson Immunoresearch Laboratories, respectively. Alexa Fluor-555 conjugated α-Bungarotoxin (α-Btx; B35451; Life Technologies) was used as a marker for acetylcholine receptors at the NMJ in skleletal muscle.
DNA constructs. GFP-tagged human LDB3-S constructs were described previously 23 . Primers are listed in Supplementary Purified LDB3 proteins. Protein expression and purification of His-tagged and untagged LDB3 peptides have been described elsewhere 9 .
RNA isolation from tissue. Total cellular RNA was isolated using TRIzol Reagent (Life Technologies) followed by on-column DNase I digestion and purification with RNeasy Mini Kit (Qiagen). RNA with greater than 8 RIN (Agilent 2100 Bioanalyzer) were used for all assays. RNA was stored at − 80 °C.

Reverse transcription: polymerase chain reaction (RT-PCR).
RT was done in 20 µl volume using 1 µg total cellular RNA and SuperScript III reverse transcriptase (Invitrogen) with oligod(T)s and random hexamers. The cDNA samples were stored at − 20 °C. The cDNA was quantified using the ssDNA OliGreen Quant-iT kit (Thermo Scientific). PCR primers flanking alternatively spliced Ldb3 exons are described in Supplemental Table 2 (Supplementary file 1). PCR products were resolved on agarose gels, stained with GreenGlo (Denville, Holliston, MA) and analyzed on a ChemiDoc XRS + Molecular Imager (Bio-Rad). These agarose gel bands were excised and isolated cDNA was either directly analyzed by Sanger sequencing or cloned into pCR2.1 plasmid by TOPO-TA cloning (Life Technologies) and sequenced from individual colonies.
Droplet digital PCR (ddPCR). ddPCR assays were run on Bio-Rad QX200 system (Bio-Rad, Hercules, CA) using TaqMan probes spanning constitutively spliced exons 2 and 3 (Mm01208763_m1; Life Technologies), thus allowing measurement of total Ldb3 transcript levels. Equal amounts of input cDNA (5 ng) were used per reaction and values are presented as copies per ng of input cDNA. Multiple biological replicates were used in each assay (n ≥ 3). At least three independent assays were performed for each sample. Data were analyzed using the QuantaSoft software (Bio-Rad). The relative LDB3 expression in mouse brain regions (mouse cortex, hippocampus, basal ganglia, and cerebellum) during postnatal development stage P0, P4 and P8 was very low/ negligible hence P10 was used as a starting reference point to compare with the adult stage (6 month) for ddPCR based quatification.
Single molecule real time sequencing and analysis. cDNA was synthesized from RNA samples with RIN values of ≥ 9.8 using the SMARTer PCR cDNA synthesis kit (634925; Clontech, Mountain View, CA) and amplified using PCR primers specific for Ldb3-L and Ldb3-S isoforms (Supplemental Table 2; Supplementary file 1). PCR products were purified using AMPure® PB magnetic beads (100-265-900; Pacific Biosciences, Menlo Park, CA) and quantified/sized on an Agilent 2100 Bioanalyzer. Approximately 500 ng of each sample was used to generate SMRTbell libraries and the polymerase-bound complexes were sequenced using a PacBio RS II and v3 SMRT Cells (100-171-800; Pacific Biosciences). Sequencing results were aligned to genomic sequence (Ensembl GRCm38.p3) by the ultra-fast universal RNA-seq aligner STAR (https:// github. com/ alexd obin/ STAR/ relea ses). The RNA-seq alignment program STAR v2.3.1 and the in-house MatchAnnot program (https:// github. com/ TomSk elly/ Match Annot) were used to map full-length non-chimeric reads to the Ldb3 gene locus (mouse reference version GRCm38 and GENCODE annotation M8). Reads with < 2 base pairs (bp) mismatch were categorized as fully matched and for the remaining mapped reads, an exclusion threshold was set at a count support of < 1%. We were able to map 24,719 Ldb3-L and 10,027 Ldb3-S full length, non-concatemer reads (average read length = 2875 bp and 963 bp, respectively). Of these reads, 89.7% and 94.3% were considered above set threshold, respectively. Ldb3 mapped reads were classified as known transcripts or novel splice variants based on GEN-CODE annotation by MatchAnnot program. Manual inspection by Integrative Genomics Viewer (http:// softw are. broad insti tute. org/ softw are/ igv/) defined the exon makeup of the Ldb3 reads.
Tissue samples and preparation. Hindlimb muscles and heart from mice were prepared by snap-freezing in liquid nitrogen-chilled isopentane. Brain and spinal cord tissues were collected from mice perfused with 4% paraformaldehyde, post-fixed in 4% paraformaldehyde overnight, and later equilibrated in 30% sucrose overnight. FFPE human motor cortex and lumbar spinal cord (L1-L2) tissues were from individuals over 18 years of age with no indicated neurological disorders. Human spinal cord whole tissue lysates (NB820-59258) were purchased from Novus Biologicals (Centennial, CO).
Immunofluorescence and immunohistochemistry. Immunofluorescence assays were done on floating 50 µm brain and spinal cord sections, and 9 µm skeletal muscle sections placed onto charged slides. Immunohistochemistry assays were done on 5 µm brain and spinal cord sections placed onto charged slides. Tissue sections were stained with primary antibody at 4 °C for 16 h, washed 5 times in phosphate-buffered saline for 2 min each and then stained with secondary antibody for 1 h at room temperature. A Leica BOND-MAX Automated Stainer (Leica Biosystems, Buffalo Grove, IL) with epitope retrieval of Citrate pH 6.0 for 20 min was used for immunohistochemistry. We examined at least 3 different dilutions of each antibody and pre-immune sera or blocking peptide when available. Antibody was blocked by incubation with LDB3 peptide at a ratio of 10:1 for www.nature.com/scientificreports/ 1 h at 4 °C before staining for signal validation. During each of the assays, negative controls were always processed following the same protocol with the omission of the primary antibody to evaluate any non-specific labeling. Immunofluorescence images were acquired using a Leica confocal microscope (TCS SP5 576 II) equipped with set of a 40 ×/NA 1.3 oil immersion Plan-Apochromat objective lens at 1024 × 1024 resolution. Tile scans were acquired (20 ×) using an Axio Scan.Z1 slide scanner (Zeiss) and stitching was done using the ZEN Blue software (Zeiss). All the image acquisition and display settings were the same between groups and comparisons were made for tissue sections stained on the same slide. The images were adjusted for appropriate size, correction of brightness, contrast, intensity and presentation in figures at resolution 300, using Adobe Photoshop CC 20.0.10 Release (Adobe systems, San Jose, CA). All images are shown with the representative scale.
Immunoblotting. Protein samples were separated by polyacrylamide gel electrophoresis and transferred to PVDF membranes (Life Technologies). After blocking in 5% low fat milk for 1 h at room temperature, the membranes were incubated with primary antibody overnight at 4 °C and then with HRP-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories) for 1 h at room temperature. The ChemiDoc XRS + Molecular Imager (Bio-Rad, Hercules, CA) was used for detection. The blots were cropped or adjusted for appropriate size using Adobe Photoshop CC 20.0.10 Release (Adobe systems, San Jose, CA) for presentation in figures. The original blots are shown in Supplementary File 1 (Supplementary Fig. 2).
Statistical analysis. Statistical analyses were done using GraphPad Prism 8. A two-way ANOVA using the Bonferroni's multiple comparison test was used to determine differences in Ldb3 expression between tissue and between age. Statistical significance was defined as p < 0.05.

Data availability
The data generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. Raw data of SMRT sequencing analysis is publically available in The National Center for Biotechnology  www.nature.com/scientificreports/